- Email: [email protected]
Regulating β-Catenin Nuclear Import with the Small GTPase Rap
Developmental Cell
Previews turnover during stress recovery. Nat. Cell Biol. 18, 1173–1184.
phagy use a scaffold protein to activate the Atg1 kinase. Mol. Cell 59, 372–381.
Grumati, P., Morozzi, G., Ho¨lper, S., Mari, M., €ller, S., Reggiori, F., Harwardt, M.I., Yan, R., Mu Heilemann, M., and Dikic, I. (2017). Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6, e25555.
Khaminets, A., Heinrich, T., Mari, M., Grumati, P., Huebner, A.K., Akutsu, M., Liebmann, L., Stolz, A., Nietzsche, S., Koch, N., et al. (2015). Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522, 354–358.
Helle, S.C.J., Feng, Q., Aebersold, M.J., Hirt, L., €ter, R.R., Vahid, A., Sirianni, A., Mostowy, S., Gru c , A., et al. (2017). Mechanical Snedeker, J.G., Sari force induces mitochondrial fission. eLife 6, e30292. Kamber, R.A., Shoemaker, C.J., and Denic, V. (2015). Receptor-bound targets of selective auto-
Lazarou, M., Sliter, D.A., Kane, L.A., Sarraf, S.A., Wang, C., Burman, J.L., Sideris, D.P., Fogel, A.I., and Youle, R.J. (2015). The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524, 309–314. Mochida, K., Oikawa, Y., Kimura, Y., Kirisako, H., Hirano, H., Ohsumi, Y., and Nakatogawa, H. (2015). Receptor-mediated selective autophagy
degrades the endoplasmic reticulum and the nucleus. Nature 522, 359–362. Rogov, V.V., Stolz, A., Ravichandran, A.C., RiosSzwed, D.O., Suzuki, H., Kniss, A., Lo¨hr, F., Wakatsuki, S., Do¨tsch, V., Dikic, I., et al. (2017). Structural and functional analysis of the GABARAP interaction motif (GIM). EMBO Rep. 18, 1382–1396. Smith, M.D., Harley, M.E., Kemp, A.J., Wills, J., Lee, M., Arends, M., von Kriegsheim, A., Behrends, C., and Wilkinson, S. (2018). CCPG1 is a non-canonical autophagy cargo receptor essential for ER-phagy and pancreatic ER proteostasis. Dev. Cell 44, this issue, 217–232.
Regulating b-Catenin Nuclear Import with the Small GTPase Rap Naoko Imamoto1,* 1Cellular Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.01.004
b-catenin acts as a key mediator of Wnt signaling by migrating into the nucleus. In this issue of Developmental Cell, Griffin et al. (2018) propose that facilitated nuclear import of b-catenin is actively regulated by the nuclear small GTPase Rap through its guanine nucleotide exchange factor, RAPGEF5. b-catenin is essential for embryonic development and also plays key roles in adults by regulating cell proliferation, for example during tumor initiation and progression. b-catenin plays critical roles both in the cytoplasm and in the nucleus. It is both a component of cell-cell adhesions at the plasma membrane and a nuclear mediator of Wnt signaling. These dual roles of b-catenin critically depend on the regulation of its nucleocytoplasmic localization (Clevers and Nusse, 2012; Fagotto, 2013). In this issue of Developmental Cell, Griffin et al. (2018) provide evidence that the nuclear small GTPase Rap regulates b-catenin nuclear import. b-catenin was the first protein shown to enter the nucleus without using the importin nuclear transport receptors (Fagotto et al., 1998; Yokoya et al., 1999). More than 20 members of the importin b family exist in mammalian cells and are linked to distinct biological processes (Kimura et al., 2017). The strongest evi-
dence for b-catenin not using any of the importin b family members as a transport carrier comes from experimental results that show that its transport does not require the small GTPase Ran. Ran specifies the directionality of transport mediated by importin b family members by regulating cargo-importin interactions. In eukaryotic cells, there is a steep RanGTP concentration gradient across the nuclear envelope; levels are high in the nucleus and low in the cytoplasm. Disruption of the RanGTP concentration gradient prevents importin b familymediated nuclear transport but does not affect the nuclear transport of b-catenin (Yokoya et al., 1999). Instead of using importin b family members, b-catenin possesses the ability to translocate through the nuclear pore complex (NPC) on its own, similar to importin b family members. b-catenin is the founding member of the Armadillo family, which is characterized by having only ARM repeats. By contrast, all importin b
family members possess only HEAT repeats. However, ARM and HEAT repeats show striking similarities: they are both tandemly arrayed amphiphilic a helices (Andrade et al., 2001). The structural properties of HEAT repeats are essential for the importin b family to function as nuclear transport receptors (Yoshimura and Hirano, 2016). HEAT repeat structures are flexible, and this flexibility plays an important role in their ability to simultaneously interact with multiple binding partners. Crystallographic studies, interaction studies, and molecular dynamics studies suggest that the structural flexibility of importin HEAT repeats allows them to regulate the interactions with their cargos via the small GTPase Ran and enables migration through the crowded space of nuclear pore channels through interaction with FG motifs on NPC components. An importin b fragment that possesses the ability to bi-directionally translocate through NPCs has structural similarity with b-catenin, suggesting an
Developmental Cell 44, January 22, 2018 ª 2018 Published by Elsevier Inc. 135
Developmental Cell
Previews overlap in regions essential for NPC translocation in both importin b and b-catenin (Lee et al., 2000). Like importin b, b-catenin was shown to bind NPC components, and its own nuclear migration is competitively inhibited by importin b (Fagotto et al., 1998; Yokoya et al., 1999). These results show that the nuclear migration of b-catenin proceeds through NPC interactions that overlap with importin b. Another ARM family protein, importin a, also migrates through the NPC on its own (Miyamoto et al., 2002). These observations suggest that ARM repeat proteins can penetrate NPCs on their own, similar to HEAT repeat proteins. On the other hand, b-catenin interacts with a variety of molecules involved in cell adhesion and with components of the Wnt signaling pathway. Furthermore, b-catenin activity is regulated differentially in response to intracellular and extracellular conditions. Therefore, it is believed that the nuclear transport of b-catenin must be regulated in a complex manner (reviewed in Fagotto, 2013). The study by Khokha’s group in this issue identifies a guanine nucleotide exchange factor for the small GTPase Rap, RAPGEF5, as a regulator of b-catenin nuclear translocation (Griffin et al., 2018). RAPGEF5 was previously identified as a candidate disease gene in a patient with congenital heart disease and heterotaxy, a disorder of left-right (LR) patterning. In this study, Khokha and colleagues investigated the requirement for RAPGEF5 in left-right development in Xenopus embryos, and they found essential roles for RAPGEF5 in the establishment of leftright organizer (LRO) and subsequent LR development. Knockdown of RAPGEF5 reduced the expression of foxj1 and xnr3, which are known targets of Wnt signaling in embryos. These findings led Khokha and colleagues to investigate the role of RAPGEF5 in b-catenin activity, which plays a central role in Wnt signaling. In the absence of Wnt signaling, b-catenin is phosphorylated and marked for degradation by a destruction complex. This maintains cytoplasmic b-catenin at a low level and thus prevents its nuclear accumulation. Upon the activation of Wnt signaling, the destruction complex is inactivated and cytoplasmic b-catenin accumulates and translocates into the nucleus to induce the transcription of Wnt target genes. Khokha and colleagues 136 Developmental Cell 44, January 22, 2018
demonstrated that activation of b-catenin by RAPGEF5 was not the result of cytoplasmic stabilization of b-catenin but resulted from events downstream of b-catenin stabilization. They showed that RAPGEF5 depletion reduced the luciferase signal of both wild-type and stabilized b-catenin in TOPFlash assays in Xenopus embryos, as well as in mouse embryonic fibroblasts (MEFs). In addition, by examining the subcellular localization of b-catenin, they demonstrated that nuclear b-catenin is significantly diminished in RAPGEF5 morphants, suggesting that RAPGEF5 is involved in the nuclear import of b-catenin. Moreover, nuclear import of stabilized b-catenin was reduced upon depletion of RAPGEF5. Furthermore, nuclear import of b-catenin in embryos treated with BIO, an inhibitor of GSK-3 (a component of the b-catenin destruction complex), was reduced upon depletion of RAPGEF5, confirming that RAPGEF5 depletion inhibits the nuclear localization of b-catenin even in the absence of the cytoplasmic degradation of b-catenin. Finally, Khokha and colleagues characterized the subcellular localization of RAPGEF5 in Xenopus embryos, mouse MEF cells, and human RPE cells and showed that RAPGEF5 is localized in the nuclei of all these cells. In addition, the small GTPases Rap1b, Rap2a, and Rap2b, which are the targets of RAPGEF5, are localized in the nucleus. Khokha and colleagues provided evidence that Rap is active in the nucleus using GFP-RBDRalGDS, a sensor for active Rap, and showed that b-catenin bound to Rap in IP studies. Based on these results, they propose a model in which RAPGEF5 maintains the GTPbound form of Rap in the nucleus and promotes the nuclear transport or nuclear retention of b-catenin. In the absence of an energy source, b-catenin would distribute equally between nucleus and cytoplasm (Yokoya et al., 1999). Thus, promoting accumulation of b-catenin in one compartment (e.g., nucleus) from the other requires an active mechanism or input of energy and a nuclear binding partner to maintain b-catenin in the nucleus. However, b-catenin is a rather abundant protein whose expression level may be higher than known potential nuclear binding partners. In light of these observations, the possibility that nuclear import of b-catenin is
actively regulated by RAPGEF5, as proposed by Khokha and colleagues (Griffin et al., 2018), provides a potential explanation for how such transport may be accomplished, although the mechanism still needs to be defined. The fact that RAPGEF5 is involved in left-right patterning during embryonic development suggests that this regulation of b-catenin nuclear import is physiologically relevant. Future work should examine the significance of this mechanism in other processed (for example, in cancer) involving Wnt signaling and Rap activity and explore the potential therapeutic relevance of b-catenin nuclear import regulation.
REFERENCES Andrade, M.A., Petosa, C., O’Donoghue, S.I., €ller, C.W., and Bork, P. (2001). Comparison of Mu ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18. Clevers, H., and Nusse, R. (2012). Wnt/b-catenin signaling and disease. Cell 149, 1192–1205. Fagotto, F. (2013). Looking beyond the Wnt pathway for the deep nature of b-catenin. EMBO Rep. 14, 422–433. €ck, U., and Gumbiner, B.M. (1998). Fagotto, F., Glu Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr. Biol. 8, 181–190. Griffin, J.N., Del Viso, F., Duncan, A.R., Robson, A., Hwang, W., Kulkarni, S., Liu, K.J., and Khokha, M.K. (2018). RAPGEF5 regulates nuclear translocation of b-catenin. Dev. Cell 44, this issue, 248–260. Kimura, M., Morinaka, Y., Imai, K., Kose, S., Horton, P., and Imamoto, N. (2017). Extensive cargo identification reveals distinct biological roles of the 12 importin pathways. Elife 6, e21184. Lee, S.J., Imamoto, N., Sakai, H., Nakagawa, A., Kose, S., Koike, M., Yamamoto, M., Kumasaka, T., Yoneda, Y., and Tsukihara, T. (2000). The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport. J. Mol. Biol. 302, 251–264. Miyamoto, Y., Hieda, M., Harreman, M.T., Fukumoto, M., Saiwaki, T., Hodel, A.E., Corbett, A.H., and Yoneda, Y. (2002). Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner. EMBO J. 21, 5833–5842. Yokoya, F., Imamoto, N., Tachibana, T., and Yoneda, Y. (1999). beta-catenin can be transported into the nucleus in a Ran-unassisted manner. Mol. Biol. Cell 10, 1119–1131. Yoshimura, S.H., and Hirano, T. (2016). HEAT repeats - versatile arrays of amphiphilic helices working in crowded environments? J. Cell Sci. 129, 3963–3970.
Previews turnover during stress recovery. Nat. Cell Biol. 18, 1173–1184.
phagy use a scaffold protein to activate the Atg1 kinase. Mol. Cell 59, 372–381.
Grumati, P., Morozzi, G., Ho¨lper, S., Mari, M., €ller, S., Reggiori, F., Harwardt, M.I., Yan, R., Mu Heilemann, M., and Dikic, I. (2017). Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6, e25555.
Khaminets, A., Heinrich, T., Mari, M., Grumati, P., Huebner, A.K., Akutsu, M., Liebmann, L., Stolz, A., Nietzsche, S., Koch, N., et al. (2015). Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522, 354–358.
Helle, S.C.J., Feng, Q., Aebersold, M.J., Hirt, L., €ter, R.R., Vahid, A., Sirianni, A., Mostowy, S., Gru c , A., et al. (2017). Mechanical Snedeker, J.G., Sari force induces mitochondrial fission. eLife 6, e30292. Kamber, R.A., Shoemaker, C.J., and Denic, V. (2015). Receptor-bound targets of selective auto-
Lazarou, M., Sliter, D.A., Kane, L.A., Sarraf, S.A., Wang, C., Burman, J.L., Sideris, D.P., Fogel, A.I., and Youle, R.J. (2015). The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524, 309–314. Mochida, K., Oikawa, Y., Kimura, Y., Kirisako, H., Hirano, H., Ohsumi, Y., and Nakatogawa, H. (2015). Receptor-mediated selective autophagy
degrades the endoplasmic reticulum and the nucleus. Nature 522, 359–362. Rogov, V.V., Stolz, A., Ravichandran, A.C., RiosSzwed, D.O., Suzuki, H., Kniss, A., Lo¨hr, F., Wakatsuki, S., Do¨tsch, V., Dikic, I., et al. (2017). Structural and functional analysis of the GABARAP interaction motif (GIM). EMBO Rep. 18, 1382–1396. Smith, M.D., Harley, M.E., Kemp, A.J., Wills, J., Lee, M., Arends, M., von Kriegsheim, A., Behrends, C., and Wilkinson, S. (2018). CCPG1 is a non-canonical autophagy cargo receptor essential for ER-phagy and pancreatic ER proteostasis. Dev. Cell 44, this issue, 217–232.
Regulating b-Catenin Nuclear Import with the Small GTPase Rap Naoko Imamoto1,* 1Cellular Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.01.004
b-catenin acts as a key mediator of Wnt signaling by migrating into the nucleus. In this issue of Developmental Cell, Griffin et al. (2018) propose that facilitated nuclear import of b-catenin is actively regulated by the nuclear small GTPase Rap through its guanine nucleotide exchange factor, RAPGEF5. b-catenin is essential for embryonic development and also plays key roles in adults by regulating cell proliferation, for example during tumor initiation and progression. b-catenin plays critical roles both in the cytoplasm and in the nucleus. It is both a component of cell-cell adhesions at the plasma membrane and a nuclear mediator of Wnt signaling. These dual roles of b-catenin critically depend on the regulation of its nucleocytoplasmic localization (Clevers and Nusse, 2012; Fagotto, 2013). In this issue of Developmental Cell, Griffin et al. (2018) provide evidence that the nuclear small GTPase Rap regulates b-catenin nuclear import. b-catenin was the first protein shown to enter the nucleus without using the importin nuclear transport receptors (Fagotto et al., 1998; Yokoya et al., 1999). More than 20 members of the importin b family exist in mammalian cells and are linked to distinct biological processes (Kimura et al., 2017). The strongest evi-
dence for b-catenin not using any of the importin b family members as a transport carrier comes from experimental results that show that its transport does not require the small GTPase Ran. Ran specifies the directionality of transport mediated by importin b family members by regulating cargo-importin interactions. In eukaryotic cells, there is a steep RanGTP concentration gradient across the nuclear envelope; levels are high in the nucleus and low in the cytoplasm. Disruption of the RanGTP concentration gradient prevents importin b familymediated nuclear transport but does not affect the nuclear transport of b-catenin (Yokoya et al., 1999). Instead of using importin b family members, b-catenin possesses the ability to translocate through the nuclear pore complex (NPC) on its own, similar to importin b family members. b-catenin is the founding member of the Armadillo family, which is characterized by having only ARM repeats. By contrast, all importin b
family members possess only HEAT repeats. However, ARM and HEAT repeats show striking similarities: they are both tandemly arrayed amphiphilic a helices (Andrade et al., 2001). The structural properties of HEAT repeats are essential for the importin b family to function as nuclear transport receptors (Yoshimura and Hirano, 2016). HEAT repeat structures are flexible, and this flexibility plays an important role in their ability to simultaneously interact with multiple binding partners. Crystallographic studies, interaction studies, and molecular dynamics studies suggest that the structural flexibility of importin HEAT repeats allows them to regulate the interactions with their cargos via the small GTPase Ran and enables migration through the crowded space of nuclear pore channels through interaction with FG motifs on NPC components. An importin b fragment that possesses the ability to bi-directionally translocate through NPCs has structural similarity with b-catenin, suggesting an
Developmental Cell 44, January 22, 2018 ª 2018 Published by Elsevier Inc. 135
Developmental Cell
Previews overlap in regions essential for NPC translocation in both importin b and b-catenin (Lee et al., 2000). Like importin b, b-catenin was shown to bind NPC components, and its own nuclear migration is competitively inhibited by importin b (Fagotto et al., 1998; Yokoya et al., 1999). These results show that the nuclear migration of b-catenin proceeds through NPC interactions that overlap with importin b. Another ARM family protein, importin a, also migrates through the NPC on its own (Miyamoto et al., 2002). These observations suggest that ARM repeat proteins can penetrate NPCs on their own, similar to HEAT repeat proteins. On the other hand, b-catenin interacts with a variety of molecules involved in cell adhesion and with components of the Wnt signaling pathway. Furthermore, b-catenin activity is regulated differentially in response to intracellular and extracellular conditions. Therefore, it is believed that the nuclear transport of b-catenin must be regulated in a complex manner (reviewed in Fagotto, 2013). The study by Khokha’s group in this issue identifies a guanine nucleotide exchange factor for the small GTPase Rap, RAPGEF5, as a regulator of b-catenin nuclear translocation (Griffin et al., 2018). RAPGEF5 was previously identified as a candidate disease gene in a patient with congenital heart disease and heterotaxy, a disorder of left-right (LR) patterning. In this study, Khokha and colleagues investigated the requirement for RAPGEF5 in left-right development in Xenopus embryos, and they found essential roles for RAPGEF5 in the establishment of leftright organizer (LRO) and subsequent LR development. Knockdown of RAPGEF5 reduced the expression of foxj1 and xnr3, which are known targets of Wnt signaling in embryos. These findings led Khokha and colleagues to investigate the role of RAPGEF5 in b-catenin activity, which plays a central role in Wnt signaling. In the absence of Wnt signaling, b-catenin is phosphorylated and marked for degradation by a destruction complex. This maintains cytoplasmic b-catenin at a low level and thus prevents its nuclear accumulation. Upon the activation of Wnt signaling, the destruction complex is inactivated and cytoplasmic b-catenin accumulates and translocates into the nucleus to induce the transcription of Wnt target genes. Khokha and colleagues 136 Developmental Cell 44, January 22, 2018
demonstrated that activation of b-catenin by RAPGEF5 was not the result of cytoplasmic stabilization of b-catenin but resulted from events downstream of b-catenin stabilization. They showed that RAPGEF5 depletion reduced the luciferase signal of both wild-type and stabilized b-catenin in TOPFlash assays in Xenopus embryos, as well as in mouse embryonic fibroblasts (MEFs). In addition, by examining the subcellular localization of b-catenin, they demonstrated that nuclear b-catenin is significantly diminished in RAPGEF5 morphants, suggesting that RAPGEF5 is involved in the nuclear import of b-catenin. Moreover, nuclear import of stabilized b-catenin was reduced upon depletion of RAPGEF5. Furthermore, nuclear import of b-catenin in embryos treated with BIO, an inhibitor of GSK-3 (a component of the b-catenin destruction complex), was reduced upon depletion of RAPGEF5, confirming that RAPGEF5 depletion inhibits the nuclear localization of b-catenin even in the absence of the cytoplasmic degradation of b-catenin. Finally, Khokha and colleagues characterized the subcellular localization of RAPGEF5 in Xenopus embryos, mouse MEF cells, and human RPE cells and showed that RAPGEF5 is localized in the nuclei of all these cells. In addition, the small GTPases Rap1b, Rap2a, and Rap2b, which are the targets of RAPGEF5, are localized in the nucleus. Khokha and colleagues provided evidence that Rap is active in the nucleus using GFP-RBDRalGDS, a sensor for active Rap, and showed that b-catenin bound to Rap in IP studies. Based on these results, they propose a model in which RAPGEF5 maintains the GTPbound form of Rap in the nucleus and promotes the nuclear transport or nuclear retention of b-catenin. In the absence of an energy source, b-catenin would distribute equally between nucleus and cytoplasm (Yokoya et al., 1999). Thus, promoting accumulation of b-catenin in one compartment (e.g., nucleus) from the other requires an active mechanism or input of energy and a nuclear binding partner to maintain b-catenin in the nucleus. However, b-catenin is a rather abundant protein whose expression level may be higher than known potential nuclear binding partners. In light of these observations, the possibility that nuclear import of b-catenin is
actively regulated by RAPGEF5, as proposed by Khokha and colleagues (Griffin et al., 2018), provides a potential explanation for how such transport may be accomplished, although the mechanism still needs to be defined. The fact that RAPGEF5 is involved in left-right patterning during embryonic development suggests that this regulation of b-catenin nuclear import is physiologically relevant. Future work should examine the significance of this mechanism in other processed (for example, in cancer) involving Wnt signaling and Rap activity and explore the potential therapeutic relevance of b-catenin nuclear import regulation.
REFERENCES Andrade, M.A., Petosa, C., O’Donoghue, S.I., €ller, C.W., and Bork, P. (2001). Comparison of Mu ARM and HEAT protein repeats. J. Mol. Biol. 309, 1–18. Clevers, H., and Nusse, R. (2012). Wnt/b-catenin signaling and disease. Cell 149, 1192–1205. Fagotto, F. (2013). Looking beyond the Wnt pathway for the deep nature of b-catenin. EMBO Rep. 14, 422–433. €ck, U., and Gumbiner, B.M. (1998). Fagotto, F., Glu Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr. Biol. 8, 181–190. Griffin, J.N., Del Viso, F., Duncan, A.R., Robson, A., Hwang, W., Kulkarni, S., Liu, K.J., and Khokha, M.K. (2018). RAPGEF5 regulates nuclear translocation of b-catenin. Dev. Cell 44, this issue, 248–260. Kimura, M., Morinaka, Y., Imai, K., Kose, S., Horton, P., and Imamoto, N. (2017). Extensive cargo identification reveals distinct biological roles of the 12 importin pathways. Elife 6, e21184. Lee, S.J., Imamoto, N., Sakai, H., Nakagawa, A., Kose, S., Koike, M., Yamamoto, M., Kumasaka, T., Yoneda, Y., and Tsukihara, T. (2000). The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport. J. Mol. Biol. 302, 251–264. Miyamoto, Y., Hieda, M., Harreman, M.T., Fukumoto, M., Saiwaki, T., Hodel, A.E., Corbett, A.H., and Yoneda, Y. (2002). Importin alpha can migrate into the nucleus in an importin beta- and Ran-independent manner. EMBO J. 21, 5833–5842. Yokoya, F., Imamoto, N., Tachibana, T., and Yoneda, Y. (1999). beta-catenin can be transported into the nucleus in a Ran-unassisted manner. Mol. Biol. Cell 10, 1119–1131. Yoshimura, S.H., and Hirano, T. (2016). HEAT repeats - versatile arrays of amphiphilic helices working in crowded environments? J. Cell Sci. 129, 3963–3970.